Bioactive compounds protection method and compositions containing the same

ABSTRACT

The present invention relates to means for protecting bioactive materials in mammalian food or feed formulations used to enhance the health status of mammals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/561,541, filed Dec. 19, 2005, which is the U.S. National Stage ofInternational Application No. PCT/IL2004/000532, filed Jun. 17, 2004,which claims the benefit of U.S. Provisional Application Nos.60/548,164, filed Mar. 1, 2004, and 60/479,860 filed Jun. 20, 2003, thecontents of each of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates generally to a method for the delivery of healthpromoting compounds to mammals and more specifically the inventionrelates to a method for encapsulating and embedding a bioactiveingredient in mammalian food or feed formulation.

BACKGROUND OF THE INVENTION

Bioactive proteins include EGF (Epidermal Growth Factor), insulin andinsulin-like growth factors, insulin-like growth factors' bindingproteins, immunoglobulins (e.g. H. Pylori antibody) proline-richpolypeptides, lactoferrin, proteases, lactalbumin, interleukin,lysozyme, TGFA (Transforming Growth Factor A) and PDGF (Platelet DerivedGrowth Factor).

These proteins may have been shown to have a positive effect on one ormore of the following health promotion advantages; protein efficiencyratios, weight gain, immune system functionality, proper growth oforgans and cells, systemic and local DNA regulation, maintenance of goodhealth, improvement of disease states and the recovery and cure fromdisease states

Most industrial processes, involve manufacturing conditions that areextremely destructive to potential viability of bioactive compounds likefunctional proteins. These include high temperatures and pressures, lowand high pH, exposure to light, desiccation and other similarly hostilemanufacturing conditions. In addition, supply chain constraints imposelonger shelf life requirements wherein extended storage under adverseconditions, cause loss of biological activity of these proteins. As aresult, inclusion of such compounds in commercially produced feeds andfoods (e.g. complete animal rations or baby formulas) is difficult.

This is crucial because mammalian neonates, like human infants, as wellas many livestock animal neonates, are frequently deprived of theirnatural mother's milk immediately or shortly after birth and arenourished primarily with milk substitutes and later, after weaning, withsemi-solid and solid mixes and pellets.

This typically means that the desired positive health benefits providedby the original natural mother's milk are largely absent from thenatural milk substitute products, as well as from the follow-on,post-weaning mixes and pellets used to feed agriculturally importantanimal infants.

For example, the immunological components of colostrum and follow-onmature milk include IgG, IgM and IgA. These components confer passiveimmunity to the neonate and protection against infection during theinitial period after parturition. It is has been shown that the presenceof insulin (and potentially IGF-1, IGF-2 and EGF) in colostrum andfollow-on mature milk leads to a faster and improved development ofmammalian neonates' gastrointestinal system fed with such natural mothermilk, compared with mammalian neonates fed with artificial substitutefoods or feed. Further, it has been demonstrated, that the presence ofinsulin in colostrum and follow-on mature milk, has a positive effect onreducing of the probability that mammalian infants will develop DiabetesType 1 (Insulin-dependent Diabetes) later in life. However, insulin andinsulin-like proteins are substantially sensitive to manufacturing andenvironmental storage conditions such as light, radiation, low/hightemperatures, low/high pressures, gastrointestinal tract digestivecompounds and the presence of chemical compounds.

SUMMARY OF THE INVENTION

In one embodiment of the invention a method is provided forencapsulating and embedding a bioactive ingredient in mammalian newbornformulation comprising the steps of (i) mixing the bioactive ingredientwith a food grade or feed grade encapsulating material so as to form aliquid blend, (ii) drying of the liquid blend so as to form a dry blend,and (iii) adding the dry blend to the mammalian newborn formulation,thereby being a method for encapsulating and embedding a bioactiveingredient in mammalian newborn formulation.

In another embodiment of the invention a method is provided forencapsulating and embedding a bioactive ingredient in mammalian newbornformulation comprising the steps of (i) mixing the bioactive ingredientwith a food grade or feed grade encapsulating material so as to form aliquid blend, (ii) drying of the liquid blend so as to form a dry blend,(iii) coating the dry blend with at least one additional food grade orfeed grade layer of encapsulating material, and (iv) adding the dryblend to the mammalian newborn formulation, thereby being a method forencapsulating and embedding a bioactive ingredient in mammalian newbornformulation.

In another embodiment of the invention, a newborn formulation isprovided comprising a bioactive ingredient being encapsulated orembedded in a food grade or feed grade encapsulating material.

In one embodiment of the invention, a method is provided for improvingthe health status, the growth and the development of a mammal comprisingthe steps of administering to the mammal a newborn formulationcomprising a bioactive ingredient being encapsulated or embedded in afood grade or feed grade encapsulating material said compound capable ofimproving the health, the growth and the development of a mammal.

In another embodiment of the invention, a method of enriching an infantformula or milk substitute is provided, comprising the step of admixinginto the infant formula or the milk substitute, a bioactive ingredientbeing encapsulated or embedded in a food grade or feed gradeencapsulating material.

In one embodiment of the invention, a method is provided forencapsulating or embedding a bioactive ingredient in mammalian solid orsemi-solid feed formulation comprising; (i) mixing the bioactiveingredient with a food grade or feed grade encapsulating materialforming a liquid blend, (ii) drying of the liquid blend forming a dryblend, (iii) coating the dry blend with at least one additional foodgrade or feed grade layer of encapsulating material, and (iv) adding thedry blend to the mammalian, solid or semi solid, feed formulation.

In another embodiment, a mammalian solid or semi-solid feed formulationis provided, wherein the solid or semi-solid formulation comprises abioactive ingredient encapsulated or embedded in a food grade or feedgrade encapsulating material.

In one embodiment of the invention, a method is provided for improvingthe health, the growth and the development of a mammal comprisingadmixing into the mammalian solid or sem-isolid feed formulationcomprising a bioactive ingredient encapsulated or embedded in a foodgrade or feed grade encapsulating material

In another embodiment of the invention, a method is provided ofenriching mammalian solid or semi-solid feed formulation, comprising thestep of admixing into the mammalian solid or semisolid feed formulationa bioactive ingredient being encapsulated or embedded in a food grade orfeed grade encapsulating material made with the method comprising; (i)mixing the bioactive ingredient with a food grade or feed gradeencapsulating material forming a liquid blend, (ii) drying of the liquidblend forming a dry blend, (iii) coating the dry blend with at least oneadditional food grade or feed grade layer of encapsulating material, and(iv) adding the dry blend to the mammalian, solid or semi solid, feedformulation.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the invention, a method is provided for theencapsulation of a bioactive material in a food grade or feed gradeglassy matrix, the method comprising; (i) mixing a homogeneous intimatemixture between at least one bioactive material and at least one wallforming, food grade or feed grade encapsulating material creating ablend, (ii) mixing said blend with an appropriate plasticizer, (iii)rapidly removing said plasticizer while inhibiting crystallization ofthe wall forming material thereby resulting in encapsulation of thebioactive material in a food grade or feed grade glassy matrix.

In another embodiment of the invention, a method is provided for theencapsulation of a bioactive material, comprising; (i) mixing at leastone bioactive material with a molten, at least one wall-forming foodgrade or feed grade encapsulating material, and (ii) rapidly cooling themolten, at least one wall forming material thereby resulting inencapsulation of the bioactive material in a food-grade or feed-gradeglassy matrix.

In the food and pharmaceutical industries, for example,microencapsulation is used to stabilize the core material, to controlthe timing and rate of the release of the core material and to separateand prevent chemical interaction between reactive or incompatiblecomponents of a multicomponent formulation. Thus, microencapsulationmakes it possible to protect sensitive food components, to ensureagainst nutritional value loss and to mask or preserve flavors andaromas. Encapsulation also increases stability of vitamin supplements,for example, which are normally sensitive to UV radiation, light,oxygen, metals, humidity and temperature. Microencapsulation is alsoutilized in the pharmaceutical industry to protect the lining of themouth and esophagus from harsh, orally administered drugs which arereleased in the stomach by the action of stomach acids on themicrocapsule coating.

Encapsulation is a process where one or more active ingredients arecoated with, or entrapped within, another material or system.Encapsulation of heat sensitive compounds, such as for examplenutraceutical components, enzymes or bioactive proteins, into matrixesthat are edible, is generally difficult for a number of reasons. Not theleast of which is that conventional encapsulation processes, whichexpose matrix material and encapsulants to high temperatures such asthose, encountered in extrusion, causes thermal destruction or loss ofbiological viability of the encapsulant. Thus, either large overdoses ofencapsulant, which would be very expensive and potentially hazardous,would be required, or the encapsulant would not sustain theencapsulation process at all. If the encapsulant can be encapsulatedinto a matrix under sufficiently low temperatures, the resulting productis a solid, that is characterized as a hard glass-like solid that iscapable of being processed further to yield a flowable powder, amenableto further processing. Moreover, although, the temperature at which theparticles are consumed, or the eating temperature, is generally lowerthan 50 degrees Celsius, which is far below the glass transitiontemperature, T.sub. carefull design of the glassy matrix can release theencapsulant under desired conditions of temperature, moisture and pH.They could also be used as dense pellets for a variety of processingapplications, where a controlled release of the heat sensitiveencapsulant is desired. The physical hardness of the products and theirmechanical stability are advantageous for many processing applications.

In one embodiment, plasticizer as used herein means an additionalcompound capable of increasing the free volume of the liquid encapsulantwithout affecting the overall cumulative volume of both encapsulatedmatrix and the plasticizing compound.

In one embodiment of the invention, protected bioactive ingredient isprovided, including, but not limited to, proteins for use in dietaryformulations.

In another embodiment of the invention, a method of manufacture theprotected bioactive ingredient is provided, so as to retain biologicalactivity of these proteins.

The invention may be used to preserve biological activity of bioactiveingredient against any of the following or similarly destructivefactors; adverse temperature, pressure, humidity, pH, osmoticconcentration, ionic concentration, chemical degradation, presence ofmetals, surfactants and chelators, radiation (including but not limitedto UV, IR, Visible light), enzymatic and microbial degradation. Inaddition, the present invention may be used to protect bioactiveingredients against physical changes including but not limited to firstand second order phase transitions.

In one embodiment of the invention, a protected bioactive ingredient isprovided, which comprises at least one protecting layer enveloping abioactive ingredient present in maternal mammalian milk.

In another embodiment of the present invention, maternal mammalian milkas used herein means colostrums.

In another embodiment of the invention, the protected bioactiveingredient is present in a natural mammalian milk but its concentrationis significantly lower, non viable, non available or non-existent incommercially processed milk or milk substitute.

In another embodiment of the present invention, concentration as usedherein means Molar concentration and its fractions, or percentagerelative to that existing in colostrums.

As used herein, the term “significantly lower” in one embodiment meansthat the amount of the bioactive ingredient in commercially processedmilk is between about 0.1 to about 50 percent of that present incolostrum.

In one embodiment of the invention, the amount of the bioactiveingredient in commercially processed milk is at the most, 50 percent ofthat present in colostrum.

In another embodiment of the invention, the amount of the bioactiveingredient in commercially processed milk is at the most, 25 percent ofthat present in colostrum.

In another embodiment of the invention, the amount of the bioactiveingredient in commercially processed milk is at the most 10 percent ofthat present in colostrum.

In another embodiment of the invention, the amount of the bioactiveingredient in commercially processed milk is at the most 1 percent ofthat present in colostrum.

In another embodiment of the invention, the amount of the bioactiveingredient in commercially processed milk is at the most 0.1 percent ofthat present in colostrum.

In another embodiment, the amount in the processed milk is notdetectable by the means known to one skilled in the art.

In one embodiment of the invention, at least a single protecting layerenable the maintenance of the bioactive properties of the bioactiveingredient while in a “dormant state”, which is, in one embodiment ofthe invention, when the protected protein is in dry or materially dryconditions, such as those present in powdered infant formulas, milksubstitute products, and semi-solid/solid mixes and pellets.

In another embodiment, dormant state of the protein as used herein meansthe preservation of the native tertiary and quarternary structures ofthe protein in an anhydrous state.

In one embodiment of the invention the at least one single protectinglayer provides protection to the encapsulated bioactive ingredient, sothat the protein shall materially maintain its bioactive properties inhostile conditions such as high temperatures normally leading toproteins' denaturation, high pressures, humidity, adverse osmoticpressures, high or low pH, strong enzymatic degradation, high solventconcentration and the like. Then, in another embodiment, based on thetriggering event, the outer protection layer is dissolved, and the“dormant” bioactive ingredient will be released and becomephysiologically active.

In one embodiment, the protected bioactive ingredient is designed in away whereby the release of the bioactive ingredient occurs beforeentering the GI system of the newborn mammal consuming the formulation.

In another embodiment of the invention, the release may be while incontact with the different parts of the gastrointestinal tract.

In one embodiment of the present invention, the encapsulated bioactiveingredient will be protected from conditions encountered duringcommercial extrusion process, including but not limited to coldextrusion or hot extrusion either at standard temperatures and pressuresor at conditions different than standard temperatures and pressures.

In another embodiment of the present invention, the encapsulatedbioactive ingredient will be protected from conditions encounteredduring commercial size reduction processes, including but not limited tocolloid mills, both stator rotor of the frusto conical type, as well ascron and tooth type, ball mills, impact mills, jet impingement mills,homogenizing mills, sonication, high velocity mixers and membraneemulsification devices.

In one embodiment of the present invention, the encapsulated bioactiveingredient will be protected from conditions encountered duringcommercial baking processes.

In another embodiment, the protected bioactive ingredient will beprotected from conditions encountered during commercial freezingprocesses.

In one embodiment of the invention, a newborn formulation is provided,comprising a bioactive ingredient being encapsulated or embedded in anedible ingredient.

In one embodiment of the invention, the newborn formulation may be aninfant formula or a milk replacer/substitute for mammal's newbornconsumption.

In another embodiment, the milk replacer/substitute as used herein isany milk replacer/substitute for mammalian neonates wherein the mammalsare of the bovine, equine, and swine families for examples calf, lamb,pig, cows, sheep, goat, yaez, cats, dogs and horses.

In one embodiment of the invention, the milk replacer/substitute is anymilk replacer/substitute for mammalian neonates wherein the mammals areof the feline and canine families.

In another embodiment the bioactive ingredient is encapsulated in aplasticizable matrix material, which is plasticizable at lowtemperatures by a liquid plasticizer or by liquid encapsulant component,which may be a plasticizable biopolymer.

In one embodiment, the plasticized material includes but is not limitedto a carbohydrate polysaccharides, such as pentosans, a physically orchemically modified starch or cyclodextrin and mixtures thereof.

In another embodiment, the plasticized material is a polymer such aspolyvinylpyrrolidone (PVP, Povidone) or other non-hydrophobic polymerssuch as N-vinylpyrrolidone (NVP) and (vinyl)acetate copolymers,(polyvinyl)alcohol chitosan or mixtures thereof. In one embodiment, theplasticized material is cellulose esters, cellulose ethers, andpolyethylene glycol. In another embodiment, the plasticized material isa hydrocolloid such as xanthan, carragenan, alginate, gum arabic, gumacacia, gum tragacanth, gum conjac and mixtures thereof.

In one embodiment, the plasticized material is glutenins and gliadins,such as vital wheat gluten or isolated gluten, zein, vegetable or dairyproteins such as protein from soy or milk, and mixtures thereof.

In another embodiment of the present invention, starches that may beused in the present invention are physically or chemically modifiedstarches, with amylose/amylopectin ratios of between about 1 to about0.001, derived from corn, wheat, rice, potato, tapioca, yuka and arrowroot.

In one embodiment, sources of starch which may be used also includeflours from grains such as corn, wheat, durum wheat, rice, barley, oat,or rye, and mixtures thereof.

In another embodiment, since the microcapsules formed are to be used ina newborn formula or in a solid or semi-solid feed formulation, onlywall material approved by the FDA or similar regulatory body in Europeand elsewhere shall be used.

In one embodiment the GRAS list provides a listing of compounds that canbe used for forming the capsule walls.

In one embodiment of the present invention, any other food-grade orfeed-grade encapsulating material, which has been approved by arecognized regulatory body for human and/or animal consumption (asapplicable), shall serve as the encapsulation material in the process.

In one embodiment of the present invention, the wall material used ispoly (DL-lactide-co-glycolide).

In another embodiment of the invention the food grade or feed-gradeencapsulating material, used in the neonate formulation comprises,polysaccharide, maltodextrin, milk powder, whey protein, lipid, gumArabic or microcrystalline cellulose or combinations thereof.

In one embodiment of the invention the bioactive ingredient beingencapsulated or embedded maintains or substantially maintains itsbiologically bioactive function and properties during the process offormulating the newborn formulation.

In one embodiment of the invention the bioactive ingredient beingencapsulated or embedded maintains or substantially maintains itsbiologically bioactive function and properties during the normalshelf-life of the underlying newborn formulation or solid or semi-solidfeed formulation in which it is incorporated.

In one embodiment of the invention the bioactive ingredient may be aglycoprotein, immunoglobulin, peptide, polypeptide, hormone or enzyme.

In another embodiment of the invention, the bioactive ingredient isinsulin, IGF-I, IGF-2, or EGF.

In one embodiment of the invention the bioactive ingredients include butis by no way limited to alpha-1-proteinase inhibitor, alkalinephosphatase, angiogenin, antithrombin III, chitinase, extracellularsuperoxide dismutase, Factor VIII, Factor IX, Factor X, fibrinogen,glucocerebrosidase, glutamate decarboxylase, human serum albumin, myelinbasic protein, lactoferrin, lactoglobulin, lysozyme, lactalbumin,proinsulin, soluble CD4, component and complexes of soluble CD4, tissueplasminogen activator and a variant thereof combinations thereof andpharmaceutically acceptable salts thereof.

In another embodiment of the present invention, the insulin or any otherproteins may be recombinant, synthetic, purified from natural source,biologically active and a peptide or polypeptide having an amino acidsequence of human or mammalian protein (e.g., human or bovine or porcineinsulin).

In one embodiment, crude extracts may be useful, depending on the methodof their manufacture, for example synthetic protein may be manufacturedusing chemical peptide synthesis or any other similraly suitable method.

In one embodiment of the invention the newborn formulation comprisesuniformly sized particles of encapsulated bioactive ingredient, whereinthe particles have a radius between about 0.001 and about 5,000micrometers.

In one embodiment of the present invention, solid feed formulation asused herein means a formulation able to maintain its density at roomtemperature and support its own weight.

In another embodiment of the invention, semi-solid formulation as usedherein means formulations capable of flowing under their own weight,with viscosities between about 1 to about 600,000 Pascal seconds.

In one embodiment of the present invention, the formulation is beingused for post weaning mammals.

In another embodiment, post-weaning mammals as used herein refers to theage at which the intensively grown mammals are typically weaned off themother's milk. For example, intensively grown lambs are typically weanedbetween 25-35 days from birth. Intensively grown piglets are typicallyweaned between 30-50 days from birth; Intestively-grown calves aretypically weaned between 40-70 days from birth.

In all of these newborn animals, in one embodiment of the invention, theprovided quantity of the milk replacer containing the bioactiveingredient is gradually reduced, and the quantity of the mix, pellets orother semi-solid or solid feed is increased.

According to another embodiment of the present invention, theintegration of a bioactive ingredient in mix/pellets was advantageousfor as long as 1-9 months post-weaning.

In one embodiment of the invention, the solid or semi-solid feedformulation may be in the form including but not limited to a mash,pellets, granules, agglomerate, extrudate or combinations thereof.

In another embodiment of the invention, the bioactive ingredient beingencapsulated or embedded maintains or substantially maintains itsbiological function during the process of formulating the mammaliansolid or semi-solid feed formulation.

In another embodiment of the invention, the bioactive ingredient beingencapsulated or embedded maintains or substantially maintains itsbiological function during the digestion of the feed.

In one embodiment of the invention, the bioactive ingredient beingencapsulated or embedded is released upon contact with a liquid.

In one embodiment of the invention, the solid or semi-solid feedformulation is protein glycoprotein, immunoglobulin, peptide,polypeptide, hormone or enzyme, either alone or in combination.

In another embodiment of the invention, the bioactive ingredient may beinsulin, IGF-I, IGF-II or EGF, either alone or in combination.

In one embodiment of the invention, the bioactive ingredient is beingencapsulated or embedded in a polysaccharide, such as maltodextrin.

In another embodiment of the invention, the mammalian solid orsemi-solid feed formulation comprises uniformly sized particles ofencapsulated bioactive ingredient, wherein the particles have an averagesize of between about 10 to about 4000 micrometers.

The formulations used in one embodiment of the invention are efficientfor increasing the rate of weight gain of mammals, preventing diarrheaand other gastric disorders and are useful for increasing the lifeexpectancy of mammals after birth.

Products containing protected bioactive ingredients according to anotherembodiment of the present invention may be consumed by a variety ofsubjects, including, but not limited to, preterm infants, post-dischargepreterm infants, term infants, babies, toddlers, children, adolescents,adults, elderly humans, the infants or adults of mammalian non-humananimals, including but not limited to bovine, porcine, caprine, feline,canine or equine species as well as infants or adults of non-mammaliananimals.

In another embodiment of the invention a method for encapsulating andembedding a bioactive ingredient in mammalian newborn formulation isprovided, comprising the steps of, (i) mixing the bioactive ingredientwith an edible food grade or feed grade encapsulating material forming aliquid blend; (ii) drying of the liquid blend; (iii) coating the dryblend with at least one additional food grade or feed gradeencapsulating material layer; and (iv) adding the dry blend to themammalian newborn formulation.

In one embodiment the mammalian newborn food formulation may be infantformula or milk replacer/substitute. Such a formulation is in anotherembodiment, a form of powder, a solution, a suspension, an emulsion, anointment, a cream in both liquid, semi-solid or a solid form

In another embodiment of the invention, a formulation for post weaningmammals which is a solid or a semi-solid formulation is provided,comprising an encapsulated and embedded bioactive ingredient prepared bythe following process: (i) mixing the bioactive ingredient with a foodgrade or feed grade encapsulating material so as to form a liquid blend;(ii) drying of the liquid blend so as to form a dry blend; (iii) coatingthe dry blend with at least one additional food grade or feed gradeencapsulating material layer; and (iv) adding the dry blend to themammalian solid or semi-solid feed formulation. The solid or semi-solidformulation may be in a form of pellets or mash/ mix.

Further, according to one embodiment of the present invention, the stepof mixing the bioactive material and the wall forming food grade or feedgrade material, involves the addition of liquid, such as, but notlimited to: water, saline, alcohol, molasses, or similar food grade orfeed grade encapsulating material solvent.

In another embodiment of the present invention, the ratio between thefood grade or feed grade material and the solvent of the food grade orfeed grade encapsulating material may be in one embodiment of theinvention between about 1:1 to about 1:1000.

In one embodiments of the invention the ratio between the food grade orfeed grade material and the solvent of the food grade or feed gradeencapsulating material is between 1:3 and 1:100.

In another embodiment of the invention, the dry blend is ground further.

The encapsulated bioactive ingredient in one embodiment may be furtherencapsulated by at least one additional protection layer, which may beformed in another embodiment of the same food grade or feed gradeencapsulating material or, in another embodiment a different food orfeed grade encapsulating material.

In one embodiment the dry blend is further mixed with said food or feedgrade encapsulating material so as to form at least one other layer offood grade or feed grade encapsulating material layer enveloping thebioactive ingredient.

In one embodiment of the invention the bioactive ingredient may bealpha-1 proteinase inhibitor, alkaline phosphatase, angiogenin,antithrombin III, chitinase, extracellular superoxide dismutase, FactorVIII, Factor IX, Factor X, fibrinogen, glucocerebrosidase, glutamatedecarboxylase, human serum albumin, myelin basic protein, lactoferrin,lactoglobulin, lysozyme, lactalbumin, proinsulin, soluble CD4, componentand complexes of soluble CD4, tissue plasminogen activator or variant,pharmaceutically acceptable salt or combination thereof.

In another embodiment of the invention, the food grade or feed gradeencapsulating material is a polysaccharide, milk powder, whey protein,lipid, gum Arabic microcrystalline cellulose, their analogs orcombinations thereof

In one embodiment of the invention the food grade or feed gradeencapsulating material, is a solid at temperatures of up to 70° C.

In another embodiment of the invention, the step of drying the foodgrade or feed grade encapsulating material and the bioactive material isdone using the methods including but not limited to; freeze drying,vacuum drying, spray drying, osmotic dehydration, fluidized beddehydration, solvent evaporation dehydration, sonication assisteddehydration, microwave-assisted dehydration, RF-assisted dehydration,either alone or commercially acceptable combinations thereof.

In one embodiment of the invention, the liquid mix is lyophilized afterincorporating at least one bioactive ingredient and at least one food orfeed grade encapsulating material ingredient.

In one embodiment lyophilization produces droplets containing at leastone protected bioactive ingredient and at least one food grade or feedgrade encapsulating material in a glassy state.

In one embodiment, a flash freezer is employed to dry the liquid mixthrough the utilization of liquid gas, including, but not limited to,nitrogen, CO₂, etc.

In one embodiment, the size of the droplets will vary between about 10and about 5,000 micrometers.

In another embodiment the droplets size distribution depends on avariety of parameters including but not limited to, freeze sprayernozzle size, liquid gas temperature, chamber temperature, mix componentsratio, mix and gas flowrates, encapsulating food grade or feed gradematerial concentration, plasticizer type and amount and freeze chamberwall geometry.

In one embodiment of the invention, the size distribution of the glassydroplets resulting from the process ranges between 50 microns and 1,000microns.

In one embodiment this treatment results in glassy frozen microdroplets, where each micro droplet contains at least one protectedbioactive ingredient, at least one food grade or feed gradeencapsulating material and the food grade or feed grade solvent.

In another embodiment once such frozen droplets are placed intemperatures above the melting temperature of the mix, the liquid mixfrom the previous phase of the process shall be reconstituted.

In one embodiment of the invention, the process further includes thefreeze-drying of a combination of at least one bioactive ingredient andat least one food grade or feed grade encapsulating material.

In another embodiment, freeze drying may be carried out on either aliquid mixture of at least one protected bioactive ingredient and atleast one food grade or feed grade encapsulating material or on frozenglassy micro droplets as described hereinabove.

In one embodiment the result of this freeze drying process is dry glassymaterial which includes at least one food grade or feed gradeencapsulating material and the at least one protected bioactiveingredient.

In another embodiment, freeze drying was performed on a liquid mixture,the result of the process was bulk dry material, porous by nature,containing a glassy matrix of the dried food-grade or feed gradeencapsulating material encapsulating the bioactive ingredient.

In one embodiment, freeze-drying was performed on the output of theflash freeze spraying process, resulting in glassy droplets, with thefood grade or feed grade encapsulating material incorporating thebioactive ingredient.

In another embodiment, low-temperature spray drying of combination of atleast one bioactive ingredient and at least one food grade or feed gradeencapsulating material was carried out.

In one embodiment, the bioactive material was dispersed in the foodgrade or feed grade encapsulating material and atomized at a maximumtemperature of 45° C.

In another embodiment, the maximum temperature is 37° C., preventingdenaturation of the bioactive ingredient. In one embodiment, spraydrying may be carried out on a liquid mixture of at least one protectedbioactive ingredient, at least one food grade or feed gradeencapsulating material and at least one chaperon-like protectingprotein, resulting in dry material which comprises the food grade orfeed grade encapsulating material and the at least one protectedbioactive ingredient.

In one embodiment of the invention, the dehydration of the food grade orfeed grade encapsulating material and the bioactive material conductedat a temperature, which is preferably below the denaturation temperatureof the bioactive ingredient, when that bioactive ingredient is protein.

In another embodiment, the dehydration of the food grade or feed gradeencapsulating material and the bioactive material is carried out attemperature below the onset temperature for the bioactive materials'degradation threshold.

In one embodiment of the invention, the dehydration process of foodgrade or feed grade encapsulating material and the bioactive material iscarried out at a maximum temperature of 50° C.

In another embodiment of the invention, the step of drying the liquidblend results in glassy freeze-dried droplets containing a bioactiveingredient and at least one food grade or feed grade encapsulatingmaterial.

In one embodiment of the invention the step of freeze-drying is precededby a step of spraying the liquid blend through an atomizer in thepresence of a liquid gas.

In one embodiment, extrusion is used as an encapsulation method in whicha core material is dispersed in a liquid mass of a bioactive ingredientand at least one food grade or feed grade encapsulating material andultimately formed into microcapsule.

In another embodiment of the invention, encapsulating or embedding thebioactive ingredient in the formulation described above involves anadditional step of premixing the blend in a small volume of themammalian newborn formulation or food grade or feed grade encapsulatingmaterial, or semi solid or solid formulation, to ensure homogeneityprior to its mixing with the whole formulation.

In one embodiment of the invention, protection processes suited for useas used herein include, but are not limited to those which produce aprotected bioactive ingredient in the form of a: powder, amicro-encapsulated powder, a nano-encapsulated powder, a liquid, amicro-emulsified liquid, a nano-emulsified liquid, a solution, amicro-emulsified solution, a nano-emulsified solution, a spread, a mash,an ointment, micro droplets, nano-droplets, tablets and solids such asfor example, pellets.

In another embodiment of the invention, the encapsulation processincludes duplex, W/O/W, O/W/O, double or multiple emulsions.

In one embodiment of the invention, the mix of at least one bioactivematerial and at least one food grade or feed grade encapsulatingmaterial and at least one surfactant selected from the group ofsurfactants having an HLB value substantially below 7 are suspended in anon-miscible, food grade or feed grade material and further mixedaffecting size reduction using methods hereinabove mentioned.

In another embodiment, the milled emulsion is further mixed with a foodgrade or feed grade material that is miscible with the food-grade orfeed grade encapsulating material and a food grade or feed gradesurfactant selected from the group of surfactants having an HLB valuesubstantially higher than 7 and further reduced in size using one of themethods hereinabove mentioned.

According to an embodiment of the invention, following formulation of abioactive ingredient, micro emulsification or nano emulsification of thebioactive ingredients is conducted.

In one embodiment, the formulated bioactive ingredient is mixed with anemulsion incorporating water, oil phase and surfactant. As a result ofsuch mixing, the bioactive ingredient's molecules are reorganized intothe dispersed phase of the emulsion.

The protection provided to the bioactive ingredients by the micro ornano emulsion in another embodiment, relates to temperature exposureprotection, and improved solubility of the bioactive ingredients withinthe food or feed with which it is integrated, following the release ofthe bioactive ingredients from its encapsulation prior to itsconsumption and/or during the digestion process.

In another embodiment, the bioactive ingredient in the nano emulsion ormicro emulsion is initially protected within the liquid micro emulsionor liquid nano emulsion.

A person holding ordinary skill in the art would readily recognize thatthis invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thehereinbelow mentioned description. It should be appreciated that variousmodifications can be made without materially changing the scope orspirit of the current invention. It should be noted that practicing theinvention is not limited to the to the applications hereinbelowmentioned and many other applications and alterations may be madewithout departing from the intended scope of the present invention Also,it is to be understood that the lexicography employed herein is for thepurpose of description and should not be taken as limiting.

In one embodiment of the invention, a method is provided for theencapsulation of a bioactive material in a food grade glassy matrix, themethod comprising; (i) mixing a homogeneous intimate mixture between atleast one bioactive material and at least one wall forming, food gradeor feed grade encapsulating material creating a blend, (ii) mixing saidblend with an appropriate plasticizer, (iii) rapidly removing saidplasticizer while inhibiting crystallization of the wall formingmaterial thereby resulting in encapsulation of the bioactive material ina food grade or feed grade glassy matrix.

In another embodiment of the invention, a method is provided for theencapsulation of a bioactive material, comprising; (i) mixing at leastone bioactive material with a molten, at least one wall-forming foodgrade or feed grade encapsulating material, and (ii) rapidly cooling themolten, at least one wall forming material thereby resulting inencapsulation of the bioactive material in a food-grade or feed-gradeglassy matrix.

In another embodiment of the invention, a method for encapsulating andembedding a bioactive ingredient in mammalian newborn formulation isprovided, the method comprising; (i) mixing the bioactive ingredientwith a food grade or feed grade encapsulating material so as to form aliquid blend, (ii) drying of the liquid blend so as to form a dry blend;(iii) coating the dry blend with at least one additional food grade orfeed grade encapsulating material layer, and (iv) adding the dry blendto the mammalian newborn formulation thereby being a method forencapsulating and embedding a bioactive ingredient in mammalian newbornformulation.

In one embodiment of the invention, a mammalian newborn formulation isprovided comprising a bioactive ingredient being encapsulated orembedded in a food grade or feed grade encapsulating material.

In another embodiment of the invention, a method for encapsulating orembedding a bioactive ingredient in mammalian solid or semi solid feedformulation is provided, comprising the steps of; (i) mixing thebioactive ingredient with a food grade or feed grade encapsulatingmaterial so as to form a liquid blend, (ii) drying of the liquid blendso as to form a dry blend, (iii) coating the dry blend with at least oneadditional food grade or feed grade encapsulating material layer, and(iv) adding the dry blend to the mammalian solid or semi solid feedformulation thereby being a method for encapsulating and embedding abioactive ingredient in mammalian solid or semi solid feed formulation.

In another embodiment of the invention, a mammalian solid or semi-solidfeed formulation is provided, comprising a bioactive ingredient beingencapsulated or embedded in a food grade or feed grade material

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the scope of the invention.

EXAMPLES Example 1 Effects of Dietary Insulin on weight gain in Bovineneonates Materials and Methods

100 Friesy and Charole' calves in ages 12-15 days from birth, were used.Each calf received a milk replacer, of up to 6 litters a day, until the37th day from birth. From the 37th day, milk replacer quantity wasreduced by 0.5 litters every 2 days while simultaneously, pelletsquantity increased, until the calf's 57th days old, when it isconsidered fully weaned. Calves in the experimental group receive 600MicroUnits/cc. of liquid milk replacer. Calves in the control groupreceive no insulin at all. Daily growth in grams per each 1 Kg. of birthweight was measured and calculated for each calf.

Results

The insulin treated group gained up to 26% more weight comparing withthe control group during the period of between days 17 and day 67 afterbirth. At the end of the study period, the average daily weight growthin grams per each Kg. of birth weight was 22.47, compared to 18.71 g/Kgbirth weight in the control group at the same time period. These resultsconfirm that insulin is an important factors contributing to the weightgain in calves at the age of between 12-15 days and 68 days from birth.

Example 2 Effects of Feed formula Enriched with Encapsulated BioactiveIngredient on Caprine Neonates' Health Status Materials and Methods

More than 1,000 Assaf specie lambs over a period of 12 months at the ageof 2-3 days from birth, were used. The growing period was divided intotwo periods: 3-28 days from birth, during which the lambs wereexclusively fed by a milk replacer; and 29-75 days from birth, duringwhich the lambs were fed by post-weaning mix/mash. Lambs in the studygroups received between 600 MicroUnits-3,000 MicroUnits of insulin/ml.of milk replacer, and between 1,600 MicroUnits-5,000 MicroUnits ofinsulin/gr. of mix/mash. The lambs in the control groups did not receiveinsulin at all.

A liquid blend, of 4 Kilograms of Maltodextrin, 40 Liters of Saline 0.9%and 100,000 IU (International Units) of insulin was blended (e.g., 25 IUinsulin per lg of Maltodextrin). The liquid blend was then freeze-dried.The product of the process was maltodextrin encapsulated insulin. 97% ofthe bioactive properties of the insulin were maintained after thefreeze-drying process. A premix of 10 Kg. was prepared from standardmilk powder and the maltodextrin encapsulated insulin. The premix wasthen blended with 740 Kg. of standard milk replacer product, to generate750 Kg. of insulin enriched commercial milk replacer.

Results

Weight gain: On average, lambs in the study groups which receivedinsulin both in the milk replacer and the mix/mash, gained between 5% to7% more weight in comparison to the control groups. Lambs in the studygroups receiving insulin only in the milk replacer (but not in themix/mash), gained between 3% to 5% more weight in comparison to thecontrol groups.

Disease incidence: The lambs in the study groups suffered between10%-25% fewer incidents of Diarrhea and/or Pneumonia, comparing withlambs in the control groups. Further, recovery of lambs suffering suchdisease states was between 5%-20% quicker in comparison with the lambsin the control groups.

Death rate: The death rate in the study groups was between 20% and 80%lower in comparison with control groups, depending on the specificstudy. For example, in one study, the insulin fed group started thestudy with 70 newly born lambs, and at marketing (e.g. 150 days afterbirth) 68 lambs survived. (2.8% death rate). In the control group,receiving no insulin, the group started with 69 newborn lambs, and atmarketing, only 61 lambs survived (e.g. 11.6% death rate). This meansthat the death rate in the insulin-fed group was lower by 74.8%comparing with the control group.

These results confirm that insulin is an important factor contributingto the weight gain and health in lambs between the ages of 2-3 days and75 days from birth. Furthermore, the result show that encapsulatedinsulin maintains biological activity of insulin during the preparationprocess of the solid feed and during the transfer in the lamb'sdigestion system.

What is claimed is:
 1. A method for encapsulating or embedding a heatsensitive bioactive ingredient in mammalian newborn food formulation ormammalian newborn feed formulation, comprising the steps of; (i) mixinga heat sensitive bioactive ingredient with an encapsulating material,wherein said encapsulating material is food-grade, feed-grade materials,either alone or in combination, with a liquid, forming a liquid blend,(ii) drying the liquid blend forming a dry blend, (iii) coating the dryblend with at least one additional encapsulating layer; (iv) mixing thedry blend with at least one additional food-grade or feed-gradematerial; and (v) adding the dry blend to the mammalian newbornformulation, wherein the steps of admixing all of the ingredients anddrying are conducted at a temperature below 50° C., such that theactivity of the heat sensitive bioactive ingredient is substantiallymaintained, thereby encapsulating or embedding the heat sensitivebioactive ingredient in a mammalian newborn formulation.
 2. The methodof claim 1, wherein the mammalian newborn food or feed is a human infantformula.
 3. The method of claim 1, wherein the mammalian newborn food orfeed is a milk replacer, a milk substitute or a combination thereof. 4.The method of claim 1, wherein the step of drying the liquid blendfurther comprises a step of grinding the dry blend.
 5. The method ofclaim 1, wherein the heat sensitive bioactive ingredient is selectedfrom the group consisting of insulin, a glycoprotein, an immunoglobulin,a peptide, a polypeptide, a hormone, an enzyme, IGF-I, IGF-II, EGFalpha-1 proteinase inhibitor, alkaline phosphatase, angiogenin,antithrombin III, chitinase, extracellular superoxide dismutase, FactorVIII, Factor IX, Factor X, fibrinogen, glucocerebrosidase, glutamatedecarboxylase, human serum albumin, myelin basic protein, lactoferrin,lactoglobulin, lysozyme, lactalbumin, proinsulin, soluble CD4, componentor complexes of soluble CD4, tissue plasminogen activator, apharmaceutically acceptable salt thereof, an analog thereof, a variantthereof or a combination thereof.
 6. The method of claim 1 wherein thestep of drying the liquid blend is performed by freeze drying, sprayfreezing, low temperature vacuum heat drying, low temperature sprayingdrying or a combination thereof.
 7. The method of claim 6, wherein saidfreeze-drying is preceded by spray-freezing.
 8. The method of claim 7,wherein the spray-freezing is preceded by extrusion.
 9. The method ofclaim 6, wherein said freeze drying, spray freezing, extrusion or acombination thereof, results in glassy droplets, containing at least onebioactive compound and at least one food-grade or feed-gradeencapsulating material, either alone or in a combination.
 10. The methodof claim 1, wherein the mammalian newborn food or feed is specificallyformulated for the consumption of the genera of primate, bovine, ovine,canine, feline, and caprine.
 11. The method of claim 1, wherein thefood-grade or feed-grade encapsulating material is polysaccharide,maltodextrin, milk powder, whey protein, lipid, gum Arabicmicrocrystalline cellulose or a combination thereof
 12. A newborn ediblefood or feed formulation comprising a heat sensitive bioactiveingredient, encapsulated by the method of claim
 1. 13. The formulationof claim 12, wherein the formulation is in a form of a powder, asolution, a spread, an ointment, a semi-solid, a solid or a combinationthereof
 14. The formulation of claim 12, wherein the bioactiveingredient being encapsulated or embedded is released upon contact witha liquid.
 15. The formulation of claim 12, comprised of uniformly sizedparticles of encapsulated bioactive ingredient, wherein the particleshave a radius between about 1 μm and 5,000 μm.
 16. A method of enrichinga human infant formula, comprising admixing a bioactive ingredient intothe human infant formula, said bioactive ingredient being encapsulatedaccording to the method of claim 1, thereby enriching the human infantformula.
 17. The method of claim 16, wherein the human infant formula isa milk replacer, a milk substitute or a combination thereof.
 18. Themethod of claim 1, wherein said at least one additional encapsulatinglayer material comprises food grade material, feed grade material eitheralone or in combination.
 19. The formulation of claim 12, wherein theformulation is a feed in form of pellets, mash, liquid, or a combinationthereof.
 20. The method of claim 1, wherein the step of coating furthercomprises a step of grinding the coated dry blend.
 21. The method ofclaim 1, wherein the formulation is a food or feed and the step ofadding the dry blend to the mammalian food or feed formulation furtherincludes premixing the blend in a small volume of the mammalian newbornfood or feed for ensuring homogeneity.
 22. The formulation of claim 12,wherein the formulation is a feed and the step of mixing comprisesadding the bioactive material as an emulsion.
 23. The formulation ofclaim 22, wherein the emulsion is a nano-emulsion, a microemulsion or acombination thereof.